DETERMINATION OF SERUM CORTISOL LEVEL BY A DIREC T ENZYME IMMUNOASSAY USING PENICILLINASE AS LABEL

Serum cortisol level was measured by an enzyme immunoassay (EIA) using the enzyme penicillinase as a label without prior extraction and purification. Polyclonal antibodies were raised against cortisol-3-ortho-carboxymethyl-oxime (cortisol-3-0-CMO) conjugated to bovine serum albumin (BSA). This antibody showed a very low cross-reactivity with structurally related steroids (3.7% for corticosterone and 4% for II-deoxycorticosterone). Standard doses were prepared in a serum sample stripped from endogenous cortisol. Danazol, 8-anilinonaphtalosulphonic acid (8-ANS) and salicylic acid were used as blocking reagents. However these reagents were not suitable in this assay. Samples were heat treated (60° C for 30 min) in order to denature the binding proteins. The assay was sensitive from 250 pg per tube covering up to 50 ng with each point having a coefficient of variation (CV) of less then 15% throughout ten successive assays. CortisoI3-0-CM 0 was conjugated to the penicillinase following a carbodiimide procedure. The formed conjugate retained almost 90% of the enzyme activity. Recoveries of exogenously added cortisol from charcoal-stripped plasma in three different ranges varied between 90-100%. Inter and intra-assay variations showed a CV of less than 12 %. The correlation coefficient was calculated as r=0.99 using our method and the results reported by a local hospital for 20 samples.